10.1: Oxidase Test

Learning Objectives

• Demonstrate an understanding of the principle behind the oxidase test, including the role of cytochrome c oxidase in the electron transport chain.
• Perform the oxidase test, including the inoculation of the test organism onto the oxidase reagent.
• Develop skills in interpreting the results of the oxidase test, including recognizing the color change indicating a positive or negative result.
• Understand the significance of the oxidase test in differentiating between bacteria that possess cytochrome c oxidase and those that do not.
• Correlate the results of the oxidase test with the biochemical characteristics and classification of microorganisms.
• Demonstrate proficiency in recording and reporting the results of the oxidase test accurately.

 

Cytochrome c Oxidase

Aerobic respiration is an O₂-requiring process that uses energy from nutrient molecules to produce ATP molecules to provide for the cell's energy needs. During aerobic respiration, the electron transport chain transfers high-energy electrons from protein to protein and uses that energy to build up a H⁺ gradient that is utilized by ATP synthase to make ATP. At the end of the aerobic electron transport chain, an enzyme transfers the electron from the electron transport chain to O₂. In some bacteria capable of aerobic respiration, that enzyme that transfers electrons to O₂, the final electron acceptor for aerobic respiration, is called cytochrome c oxidase. (Fig.1.)

 

Figure 1: The bacterial electron transport chain is a series of protein complexes, electron carriers, and ion pumps that are used to pump H⁺ out of the bacterial cytoplasm into the extracellular space. The last enzyme in the electron transport chain can be (depending on the species) cytochrome c oxidase. The role cytochrome c oxidase plays is to remove electrons from the chain by transferring them to O₂ with H⁺ to produce water. In aerobic bacterial species that do not have cytochrome c oxidase, this process still occurs, but using an enzyme that has a different structure and different name than cytochrome c oxidase. The gradient of H⁺ produced by the electron transport chain is used by the ATP synthase to make ATP. H⁺ flows back down the electrochemical gradient into the bacterial cytoplasm through ATP synthase, providing the energy for ATP production by oxidative phosphorylation (credit: modification of work by Klaus Hoffmeier).

 

A test called the oxidase test can be used in the laboratory to determine if a bacterial species has cytochrome c oxidase. Those species that have cytochrome c oxidase are called oxidase positive and those species that do not have cytochrome c oxidase are called oxidase negative. The ability of a bacterial species to produce cytochrome c oxidase is coded in its DNA. If the cytochrome c oxidase gene is present in the bacterial species, it will produce this enzyme. If the gene is absent, that bacterial species cannot produce cytochrome c oxidase. The oxidase test is therefore useful for characterizing bacterial species and differentiating bacterial species from each other for identification purposes. For example, the gram-negative opportunist Pseudomonas aeruginosa and the gram-negative cholera-causing Vibrio cholerae use cytochrome c oxidase, which can be detected by the oxidase test, whereas other gram-negative Enterobacteriaceae, like E. coli, are negative for this test because they produce different cytochrome oxidase types.

 

Note

Bacterial species that contain cytochrome c oxidase are capable of aerobic respiration. Oxidase-positive bacterial species are all aerobic.

However, oxidase-negative species are not necessarily anaerobic. Oxidase-negative species may still be aerobic species or facultative anaerobes, but they produce and use a different enzyme, other than cytochrome c oxidase, to transfer electrons from the electron transport chain to O₂.

 

The Oxidase Test

The oxidase test is a key test to differentiate between the bacterial families Pseudomonadaceae (oxidase positive species) and Enterobacteriaceae (oxidase negative species).

The oxidase test utilizes a special reagent called an oxidase reagent. This reagent is colorless. However, in the presence of cytochrome c oxidase, the oxidase reagent will transfer its electrons to cytochrome c oxidase and exhibit a bluish or purplish color in 30 seconds or less. (Fig.2.)

• oxidase-positive bacteria contain cytochrome c oxidase and produce a change in color of the reagent from colorless to bluish or purplish in less than 30 seconds.
• oxidase-negative bacteria do not contain cytochrome c oxidase and do not change the color of the oxidase reagent in less than 30 seconds.

 

Figure 2: An oxidase test strip containing oxidase reagent is still white indicating bacteria is oxidase negative (left). The oxidase test strip containing the oxidase reagent is purple indicating the bacteria are oxidase-positive (right). 

 

Laboratory Instructions

 

Oxidase Test

1. Obtain an oxidase DrySlide.
2. Using a toothpick, add a sample of each species into separate panels of the DrySlide.
3. Start a timer for 30 seconds.
4. Watch for purple color to develop on the oxidase test strip within 30 seconds.
   • purple or bluish color change in less than 30 seconds is oxidase positive
   • no color change within 30 seconds is oxidase negative (color change after 30 seconds is considered oxidase negative)
5. Repeat for each bacterial species you are testing.

 

Attributions

• "Microbiology Laboratory Manual: Labs, 1.19 Cytochrome C Oxidase" by Dr. Rosanna Hartline, West Hills College Lemoore, LibreTexts: Biology is licensed under CC BY-NC 4.0
• Chapter Image: Red Mountain Microbiology by Jill Raymond Ph.D.; Graham Boorse, Ph.D.; and Anne Mason M.S. is licensed under CC BY-NC 4.0
• MB352 General Microbiology Laboratory 2021 (Lee) by [e-mail] is licensed under CC BY-NC-SA 4.0
• Microbiology by OpenStax is licensed under CC BY 4.0
• Red Mountain Microbiology by Jill Raymond Ph.D.; Graham Boorse, Ph.D.; Anne Mason M.S. is licensed under CC BY-NC 4.0